This work represents a continuation of our investigation into the biosynthetic mechanisms responsible for the production of met5-enkephalin in adrenal medullary chromaffin granules. We have purifieda trypsin-like adrenomedullary protease capable of generating met5-enkephalin from endogenous precursors 1000 fold using affinity chromatography in combination with gel filtration. The enzyme has an apparent molecular weight of 20,000 by gel filtration. The reactivity of the adrenal enzyme toward fluorogenic substrates and enkephalin-containing peptides was studied. The results indicate that the fluorogenic compounds are poor substrates for the enzyme; however, Peptides E and F (endogenous adrenal opioids each containing two enkephalin sequences) were cleaved. When the enzymatic products of these peptides were identified by HPLC, it was found that cleavage occurred between or after pairs of basic residues. The 8.6 Kdal N-terminal fragment of proenkephalin (which contains a carboxyl terminal met-enkephalin) and [3H]Beta-lipotropin were also used as substrates for this enzyme. While the 8.6 Kdal fragment was cleaved to form met 5-enkephalin and lys1-met5-enkephalin, no cleavage of [3H]Beta-lipotropin was detectable. We have recently begun biosynthetic studies on the octapeptide met5-enk-arg6-gly7-leu8. We have developed a sensitive radioimmunoassay for this peptide, and examined its distribution and molecular heterogeneity in adrenal and brain tissue. The brain distribution of the octapeptide was found to closely parallel the distribution of met5-enkephalin; however, in several brain areas, octopeptide immunoreactivity was heterogeneous with respect to molecular weight. Chromaffin granules contained mainly high molecular weight forms of met5-enk-arg6-gly7-leu8. Whether these high molecular weight forms function as precursors for octapeptide formation will be examined in future studies.